1. Field of Invention
The present invention relates to metabolically engineered Escherichia coli (“E. coli”), and, more specifically, to metabolically engineered E. coli for enhanced production of sialic acid.
2. Description of Prior Art
Sialic acid is a biologically complex carbohydrate that plays important roles in cell signaling, bacterial pathogenesis and tumour metastasis. The physiochemical properties of sialic acid have made it a valuable resource with an increasing demand in both medicine and biotechnology.
More precisely, sialic acid is a general term used to describe a large family of acidic sugars that are predominantly found on the cell surface of eukaryotic cells. The most common sialic acid is N-acetylneuraminic acid or Neu5Ac, an acidic nine-carbon sugar that undergoes several modifications to generate the members of the sialic acid family. As seen in FIG. 1, the diversity of the sialic acid family is represented with over 50 known members. Sialic acid represents a large family of cell-surface carbohydrates that are derived from an acidic, nine-carbon parent compound called N-acetylneuraminic acid or Neu5Ac. Neu5Ac is often decorated with acetyl, phosphate, methyl, sulfate and lactyl groups, which are required for desirable cell signaling and cell adhesion events mediated by sialic acid.
Sialic acid performs a diverse array of functions including important biological processes such as glycoprotein stability, cellular immunity, solute transport, self-recognition, neuronal plasticity and cell signaling. Sialic acid also has a significant role in human disease. For example, cancerous cells have been shown to have elevated sialic acid levels or sialylation on their surfaces. Excess sialylation has been shown to promote invasiveness and reduce intercellular interactions, which are features of tumour metastasis. Sialic acid is currently being researched as a tool for the non-invasive imaging of cancer tissue in vivo. Increased levels of sialic acid have been observed for cardiovascular disease, alcoholism and diabetes, thus making sialic acid a marker for diagnosing and detecting such deadly diseases. Some bacterial pathogens possess a polysialic acid capsule that serves as a masking agent by mimicking the extracellular surface of mammalian cells. Polysialic acid capsules protect the underlying bacteria from the host immune response, which allows the bacteria to establish an infection. Polysialic acid capsulated bacteria are leading causative agents of meningitis, a life threatening disease in adults and children. Lastly, analogs of sialic acid have found important roles in the pharmaceutical industry as influenza neuraminidase inhibitors, e.g., Relenza®, stabilizers of therapeutics and diagnostic agents.
The resource availability of sialic acid is limited and costly. Traditionally, sialic acid has been isolated from egg yolk, milk whey and edible bird nest, which requires a lengthy, cumbersome purification process. The overall yields are typically low (˜10-20%) and the purity is marginal. Synthetic routes in sialic acid chemistry are challenging, because of the target molecule's complexity. To circumvent the difficulties associated with sialic acid isolation, purification and synthetic chemistry, enzymatic synthesis is currently the preferred method for sialic acid production. The cloning and subsequent isolation of enzymes involved in the biosynthesis and degradation of sialic acid has enabled researchers to produce sialic acid in vitro by a straightforward enzymatic reaction. Enzymatic synthesis of sialic acid has a high yield (80%) and generates a pure product through a direct crystallization of the enzymatic mixture. The major drawbacks of enzymatic synthesis in sialic acid production are its cost and scalability at the industrial level.
The methods for producing sialic acid, discussed supra, are insufficient in meeting the large demand of the medical and biotechnological industries. A metabolic engineering approach that successfully overcomes the problems associated with, e.g. sialic acid isolation, purification, synthetic chemistry, production yield, cost, and scalability at the industrial level, would represent a significant and long awaited advance in the field.